Endotoxemia (also called septic shock and septic syndrome) is generally considered to result from an inability of the host defense mechanisms to cope with foreign organisms, including gram positive and negative bacteria, viruses, fungi and parasites. The majority of endotoxemia cases are caused by gram negative bacteria (Glauser et al., 1991, Lancet 338 (September) 732-736), in particular a product of gram negative bacteria called endotoxin or lipopolysaccharide (LPS), which is a component of the bacterial outer cell wall.
Because the main initiator of endotoxemia is endotoxin, many tests have been developed to measure these molecules in fluids, including the Limulus Amoebocyte Lysate (LAL) test (Cohen J., 2000, Intensive Care Med. 26: S51-S56), rabbit pyrogen test, and lethality in mice and chicken embryos (Hurley J C. Clin Microbiol Reviews 8(2): 268-292). However, measurement of endotoxin in fluids, especially blood, is a poor predictor of clinical outcome in endotoxemia for a number of reasons:                The levels of endotoxin required to trigger the biological cascade of events leading to endotoxemia varies widely from patient to patient.        The bioavailability of endotoxin varies from patient to patient depending upon the body's ability to detoxify or neutralize it.        Some patients develop sensitivity to endotoxin, or are tolerant to endotoxin.        Various biological fluids (and even fluid containers) contain agents that bind endotoxin that are capable of enhancing or limiting the biological effect of endotoxin, or can interfere with the measurement of endotoxin.        Some endotoxins are more potent than others.        The specificity and sensitivity of the LAL assay is at its limit when used for assaying endotoxin in blood or serum.        
For these reasons, efforts have been made to develop assays for the determination of the biological effects of endotoxin—as a means of determining clinical outcome—including the measurement of molecules such as Tumor Necrosis Factor (TNF), C3a, C5a, Factor XII (Hageman Factor), interleukin-1 (IL-1), γ-interferon and various other cytokines, and the measurement of the level of activation of leukocytes. Such measurements have contributed to a “sepsis score” concept developed by Casey et al. (1993, Ann Intern Med. 119: 771-778).
However, none of these tests have been sufficiently sensitive, specific or practical enough to be used in routine clinical practice. In addition, the efficacy of available treatments also limits the practical use of such prognostic and monitoring tests.
Despite this, there are a number of features of endotoxemia that make early detection, monitoring, determination of clinical outcome, prognosis determination, early intervention and informed management of affected animals clinically and economically important, viz:                Many and varied conditions can lead to endotoxemia.        Endotoxemia can lead to many other conditions.        Endotoxemia is often a peracute condition causing death if not correctly managed.        It is estimated that 20-30% of human patients in intensive care units in the USA are affected and that more than 100,000 humans die each year in the USA alone (Young L & Glauser M (Eds) Gram negative septicemia and septic shock. WB Saunders Philadelphia (1991); Parrillo J E. 1990, Ann Intern Med. 113: 227-242).        The extent of the condition in less developed countries is likely to be far higher due to poor hygiene and medical infrastructure.        In up to 50% of cases an etiological agent is not determined.        The condition is most common in hospitals in patients with other underlying diseases.        The extent of subclinical disease and its effects on human health, animal husbandry, athletic performance, and ethical management are not known.        
Apart from the direct detection of endotoxin, there have been many efforts to use secondary indicators of sepsis to diagnose and monitor this condition, including measuring heart rate, temperature, respiratory rate, cardiac output, systemic vascular resistance, plasma IL-6 levels, macrophage inflammatory protein-2, chemokine KC, protein C and C-reactive protein (Panacek et al., 2004, Crit Care Med. 32: 2173-2182; Ulloa and Tracey, 2005, Trends Mol Med. 1:56-63).
Currently no panel of biomarkers is used to define sepsis in humans (Buras et al., 2006, Nature Reviews Drug Discovery, 4: 854-865). However, a cytokine profile has been suggested (Ulloa et al., 2005, Trends Mol Biol. 11: 56-63) Reasons for this are the complexity of the disease, difficulty in defining the stage of disease and the apparent existence of two distinct but not mutually exclusive phases of inflammatory and anti-inflammatory responses (Bone R C., 1996, Crit Care Med. 24: 1125-1128).
Given the current limitations of diagnostic, monitoring and prognostic procedures for endotoxemia, especially in sub-clinical or early-stages, there is a need for more effective modalities for early detection, diagnosis, monitoring, prognosis and management of the various phases of sepsis including, acute, peracute, early stage, advanced, and sub-clinical endotoxemia.
An example of a complication arising from endotoxemia is laminitis that causes profound lameness in hoofed animals. It occurs in perissodactyl and artiodactyl animals, including horses, cattle, goats, sheep and other hoofed animals (ungulates). It is believed the condition results from the action of endotoxin on tissues and the lamellae of the inner hoof wall. Failure of the lamellae results in separation of the inner hoof capsule from the pedal bone and the subsequent (weight-bearing) driving of the pedal bone through the hoof capsule, and crushing of the corium, sole and coronet (Sloet van Oldruitenborgh-Oosterbaan M M., 1999, Vet Quarterly 21(4) 121-127).
There are a number of features of laminitis that make early detection, monitoring, early intervention and informed management of affected animals clinically and economically important, viz:                The exact cause of laminitis is not known.        The extent of subclinical disease (often called the developmental stage; Hood D M., 1999, Vet Clin Nth Amer Eq Pract 15(2): 287-294) and its effects on animal husbandry, athletic performance, and ethical management are not known.        The first 72 hours of the disease (developmental and acute stages) is the most critical period for monitoring. Animals that have not suffered major mechanical or structural failures at this stage are likely to recover.        The pathogenesis of the disease is poorly understood.        Laminitis is the largest killer of horses worldwide, usually as a result of euthanasia due to progressive disease that causes serious disability and pain.        Present diagnostics are only partially effective once the disease is established, by which time preventative management or ameliorating therapies have little effect.        There are few practical interventions available.        
Thus, there is a need for more effective modalities for early diagnosis, diagnosis of mild or sub-clinical laminitis, in the detection of specific immune responses as part of active or progressive disease, and in monitoring animals clinically affected by laminitis. Such modalities would enable better treatment and management decisions to be made in clinically and sub-clinically affected animals prior to irreversible tissue damage.
Existing technology for diagnosing endotoxemia or for monitoring conditions that lead to endotoxemia or for evaluating sequelae of endotoxemia, is limited in that the detection of bacterial endotoxin in body fluids does not correlate well with clinical signs, and the sensitivity and specificity of these technologies is insufficient to be clinically useful. In addition, because the conditions are often peracute, advanced and irreversible tissue damage may have occurred (and possibly death) by the time endotoxin is able to be detected.
In addition, existing technologies for diagnosis or evaluation of laminitis are limited and are almost entirely reliant upon clinical evaluation and the detection of lameness. In many instances the lameness can be very subtle or sub-clinical. In addition, many of these clinical changes can only be observed in advanced stages of disease, at which time irreversible tissue damage has occurred, and where humane euthanasia is the only recourse.